1. Field of the Invention
The present invention relates to novel unsymmetrical cyanine dyes and to methods of performing nucleic acid analysis in the presence of such dyes. More specifically, the present invention relates to novel unsymmetrical cyanine dyes that have high affinity to double-stranded nucleic acids acid that do not inhibit amplification reactions, particularly the polymerase chain reaction (PCR).
2. Description of Related Art
The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. PCR is perhaps the most well-known of a number of different amplification techniques.
PCR is a powerful technique for amplifying short sections of DNA. With PCR, one can quickly produce millions of copies of DNA starting from a single template DNA molecule. PCR includes a three-phase temperature cycle of denaturation of DNA into single strands, annealing of primers to the denatured strands, and extension of the primers by a thermostable DNA polymerase enzyme. This cycle is repeated so that there are enough copies to be detected and analyzed. In principle, each cycle of PCR could double the number of copies. In practice, the multiplication achieved after each cycle is always less than 2. Furthermore, as PCR cycling continues, the buildup of amplified DNA products eventually ceases as the concentrations of required reactants diminish. For general details concerning PCR, see Sambrook and Russell, Molecular Cloning—A Laboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2005) and PCR Protocols A Guide to Methods and Applications, M. A. Innis et al., eds., Academic Press Inc. San Diego, Calif. (1990).
Real-time PCR refers to a growing set of techniques in which one measures the buildup of amplified DNA products as the reaction progresses, typically once per PCR cycle. Monitoring the accumulation of products over time allows one to determine the efficiency of the reaction, as well as to estimate the initial concentration of DNA template molecules. For general details concerning real-time PCR, see Real-Time PCR: An Essential Guide, K. Edwards et al., eds., Horizon Bioscience, Norwich, U.K. (2004).
Melt curve analysis is an important technique for analyzing nucleic acids. In this method, a double stranded nucleic acid is denatured in the presence of a dye that indicates whether the two strands are bound or not. Examples of such indicator dyes include non-specific binding dyes such as SYBR® Green I, whose fluorescence efficiency depends strongly on whether it is bound to double stranded DNA. As the temperature of the mixture is raised, a reduction in fluorescence from the dye indicates that the nucleic acid molecule has melted, i.e., unzipped, partially or completely. Thus, by measuring the dye fluorescence as a function of temperature, information is gained regarding the length of the duplex, the GC content or even the exact sequence. See, for example, Ririe et al. (Anal Biochem 245:154-160, 1997), Wittwer et al. (Clin Chem 49:853-860, 2003), Liew et al. (Clin Chem 50:1156-1164 (2004), Herrmann et al. (Clin Chem 52:494-503, 2006), Knapp et al. (U.S. Patent Application Publication No. 2002/0197630), Wittwer et al. (U.S. Patent Application Publication No. 2005/0233335), Wittwer et al. (U.S. Patent Application Publication No. 2006/0019253), Wittwer et al. (U.S. Patent Application Publication No. 2007/0020672) and Sundberg et al. (U.S. Patent Application Publication No. 2007/0026421).
Unsymmetrical cyanine dyes are a group of dyes which have been used in nucleic acid and protein staining. Additional unsymmetrical cyanine dyes have been developed which have been used for binding to RNA and/or DNA. See, for example, Lee et al. (U.S. Pat. No. 4,883,867), Lee et al. (U.S. Pat. No. 4,937,198), Yue et al. (U.S. Pat. No. 5,321,130), Wittwer et al. (U.S. Patent Application Publication No. 2006/0019253), Wittwer et al. (U.S. Patent Application Publication No. 2007/0020672), Haugland et al. (U.S. Pat. No. 5,436,134), Yamamoto et al. (U.S. Pat. No. 5,563,070), Yue et al. (U.S. Pat. No. 5,658,751), Mizukami et al. (U.S. Pat. No. 6,004,816), Bieniarz et al. (U.S. Pat. No. 6,015,902), Glazer et al. (U.S. Pat. No. 6,054,272), Lee et al. (U.S. Pat. No. 6,080,868), Kubista et al. (U.S. Pat. No. 6,329,144), Deka et al. (U.S. Pat. No. 6,368,864) and Haugland et al. (U.S. Pat. No. 6,664,047).
Several problems exist with known unsymmetrical cyanine dyes. For example, certain unsymmetrical cyanine dyes can poison the PCR reaction. Other unsymmetrical cyanine dyes suffer from weak binding to nucleic acids. Still other unsymmetrical cyanine dyes both poison the PCR reaction and weakly bind to nucleic acids. These and other drawbacks of unsymmetrical cyanine dyes currently exit.